With world population expanding at a logarithmic rate, the planet will reach approximately 10 billion people in about 40 years. The demand for more contraceptive options, especially targeting the male, thus is also expected to rise. To identify new targets for male contraception, studies must be undertaken to understand critical events that sperm must undergo to become functional gametes. With this goal in mind, we propose to study the annulus, a macromolecular septin structure located on the sperm tail. Sperm that are produced without an annulus are otherwise normal structurally, but they fail to swim and cannot undergo capacitation. This condition has been shown in the laboratory with septin 4 knockout mice as well as clinically in a population of infertile men. Together, these data establish that the annulus plays a vital role in sperm function, but its mechanisms of action are unknown. We propose to isolate the annulus and determine its proteome using mass spectrometry. Both low and high stringency conditions will be used to isolate the annulus to identify as many annulus-associating proteins as possible. We will isolate the annulus at four different stages of sperm function (immotile, motile, hypermotile, and fertilization competent). Identified annulus and annulus-associated proteins will be confirmed by immunofluorescence. The identification of proteins that alter their protein tyrosine phosphorylation will also be done. From the information gathered, we will develop hypotheses about how the annulus is functioning and test them in wild-type sperm by inhibiting the proposed function and observing the effect, as well as rescue experiments in sept4-/- sperm to see if function can be restored. Understanding the vital role the annulus plays in sperm function will advance our comprehension of how these mechanisms work together to make a sperm cell become a functional gamete. This information may lead to new therapeutics for infertile men as well as novel post- testicular male contraceptives. PUBLIC HEALTH RELEVANCE: The goal of this project is to isolate the sperm annulus and determine its proteome. This data will advance the fields of septin, contraceptive, and reproductive biology. Putative new therapies for infertility as well as novel male contraceptive targets may arise from this work.